Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device. ..more

Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device.

Expected Outcome: Non-selective STK33 inhibitors will also inhibit PKA while selective ones will not. IC50s for compounds of interest are determined based on %conversion vs compound concentration and are used to decide which compounds are selective for STK33 or inhibit both kinases.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (1)(Max_Concentration).

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_SCORE:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MWhen the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

DATA AGGREGATION:Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:a. '2' (active) when all test outcomes were '2' (active), orb. '1' (inactive) when all test outcomes were '1' (inactive), orc. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.